Stable injectable oil-pectin therapeutic compositions



Patented Aug. 15, 1950 UNITED STABLE INJECTABLE OIL-PECTIN THERA- PEUTICCOMPOSITIONS Henry Welch, Silver Spring, Md., assignor to the UnitedStates of America.

No Drawing. Application August 25, 1947, Serial No. 770,558

(Granted under the act of March 3,1883, as amended April 30, 1928; 3700. G. 757) 4 Claims.

My invention relates to a novel pectin preparation and novel process forproduction thereof.

This application is a continuation in part of application Serial No.752,298, filed June 3, 1947, now Patent No. 2,491,537. In thatapplication I described a number of pectin preparations suitable forprolonging the effective therapeutic blood level concentration;

As there described, many expediencies have been proposed directed to theend of maintaining an effective therapeutic penicillin blood levelconcentration for a prolonged period of time. These include continuousintravenous injections, continuous intramuscular administrations, theestablishment of an excretory blockade in the patient by thesimultaneous administration of paraaminohippuric acid or diodrast andthe use of ice packs at the site of intramuscula injection. Prolongationof therapeutic activity has also been attempted with various slowlyabsorbed vehicles, such as peanut oil and beeswax, globin and the like.

With certain of these vehicles, such as globin and the like, prolongedaction of penicillin in the blood is not obtained to a sufiicientdegree.

The development of penicillin in oil and wax overcame the rapidabsorption of penicillin but manifested other disadvantages. Whenpenicillin is suspended as a dry salt (e. g., calcium salt) in oil withbeeswax and injected into the body, a mass is formed in the tissuesconsisting of beeswax in which penicillin is entrapped. Water from thetissues gradually dissolves out the penicillin from this beeswax matrixand in a matter of 20 to 24 hours the penicillin has been completelyabsorbed, although the beeswax itself remains for some time in thetissue.

Beeswax is a well-known sensitizer and thus produces allergic reactionswhen injected into the body in combination with peanut oil andpenicillin. Because of its structure (1. e., insolubility in tissuefluids), beeswax is absorbed with great difllculty by the human bodyrequiring on an average from twenty-five to thirty days to beassimilated by the body. Furthermore, sterile abscesses are frequentlyformed by beeswax prep- -arations and moreover are painful at the siteof injection for prolonged periods. There is,

therefore, need for a preparation which will pro- 2 long the activity ofpenicillin in the body while avoiding the disadvantages of the beeswaxpreparation.

I have discovered that some hydrophilic substances, particularly pectin,which is readily digested and assimilated by the body, form a, gellikestructure on contact with water in the body tissue. If a therapeuticproduct such as penicillin is injected intramuscularly with ahydrophiiic substance such as pectin, the colloidal gel formed by thepectin entraps the penicillin. As furthe water reaches the colloidalgel, it is slowly dissolved resulting in a corresponding slow release ofthe penicillin.

Clinical data of the product described in the above identifiedapplication and made in accord ance with the process described has shownthat desired blood levels were secured for at least a twenty-four hourperiod and that there was no toxic eifects.

Essentially one of the products described comprised a mixture of drypectin and penicillin suspended in an oil. Such products are bottled andreach the physician who is to administer it a considerable time, usuallymany months, after manufacture. The physician prefers that it beinstantly ready for injection without any mechanical manipulations.

It is accordingly desirable that any such product should besubstantially stable, 1. e., that the dry ingredients should remain insuspension in the oil. I have discovered that such stability can besecured by a control of the viscosity of the mix including a control ofthe particle size of the pectin and penicillin. When this particle sizeis sufficiently small, these dry ingredients tend to remain insuspension rathe than settling.

In general, my invention contemplates a stable mix of the productsdescribed in the above identifled application by utilizing an oilcarrier having a predetermined viscosity. Thus if peanut oil is used, Iadd in addition thereto a small percentage of acid-free hydrogenatedpeanut oil.

Inasmuch as the .essence of the invention resides in the avoidance ofthe painful effects of the beeswax preparations heretofore used, caremust be used in the hydrogenated oil employed. It must be acid free andused in limited proportions since it otherwise will produce the samepainful results as was experienced with the beeswax preparations.

Accordingly objects of my invention are .to provide a novel stablehydrophilic (pectin) preparation and a novel process for producing thesame; to provide a stable pectin product and process therefor whichprolongs therapeutic blood levels and to provide micron particle mixesof pectin, in an oil solution of a desired viscosity.

These and other objects will be clear from the detailed descriptionwhich follows.

As described in the parent application, the preferred embodiment of theh drophilic agent employed is preferably pectin. This is mixed in thedry state using particles which have a-siz'e of approximately 50 micronswith dry penicillin in a vegetable oil and a hydrogenated vegetable ofthe preparation is carried in a humidity controlled room where it ispossible to maintain a relative humidity on the order of Thus thevegetable oil used, the hydrogenated oil used, and the penicillin usedall must be so treated as to contain 1% moisture or less. As statedabove, in order to secure stability, i. e., prevent settling of thepectin or penicillin, it is desirable that these ingredients be of thesmallest practical particle size without of course destroying theirnecessary properties.

Inasmuch as the dry pectin after suspension in oil with penicillin is tobe injected into the muscle or subcutaneously, the particle size of thepectin and penicillin must be such that it will pass through a 20-gaugeneedle without difllculty. So far as the pectin is concerned, it maythus be of any size under that which will pass through a 20-gaugeneedle, but the size of the penicillin is more critical and besides theupper limit governed by the needle bore, it has been found that thesmaller the penicillin particles, the less efficacious it is inprolonging blood concentrations following injection.

It has been found that when the penicillin particle size is on the orderof from 50 to 100 microns, the most eflicient results are obtained.Penicillin of this particle size is obtained by grinding in a suitablemill and passing through a 140 mesh screen. There may a few particles aslarge as 300 to 400 microns in a batch. However, these do not causeplugging of a 20-gauge needle. Indeed, a larger bore needle, i. e., 19,18, or 17 gauge, may be used for injection and thus obviate anydifliculty even with particles of penicillin 400 microns in diameter.

The pectin is also ground ina mill to the desired size. As alreadystated, when pectin of particle size of approximately 50 microns isused. shelf tests show that some settling of the pectin takes placewithin a relatively short time.

I have found that this settling can be avoided by utilizin pectin of finparticle size. Such pectin is obtained by lowering the temperature ofpectin until it becomes brittle and grinding the pectin in thiscondition. To this end I mix the pectin with small pieces (A inch) ofDry Ice and chill it for to of an hour. When the pectin has thus becomebrittle, I grind it in a hammer mill and pass the ground product througha 250 mesh screen utilizing only that material which passes the screen.The carbon dioxide gas is eliminated by the process of drying the pectinin a vacuum oven at a temperature of 60 C. to 100 0., being careful notto overheat the pectin which would thus cause the loss of some of itsessential properties here.

The settling of the pectin is further overcome by increasing theviscosity of the vegetable oil by addition to it of hydrogenated peanutoil or other hydrogenated oils. Such hydrogenated oil, however, must bepure and contain relatively no free acids (less than .05%) since thepresence of such acids cause considerable pain to the patient oninjection. As a matter-of fact, the vegetable oil itself must also besimilarly relatively free from such free acids.

Another process for preventing settling of the pectin entails use ofextremely small particles (as small as from 0.1 to 0.5 micron averagingabout 1 micron) of pectin without the necessary addition of hydrogenatedoil. In the latter case the pectin particles are so minute that thenormal viscosity of the oil overcomes the pull of gravity on them. Thisprocedure is carried out as follows:

Twenty grams of pectin is dissolved in 400 cc. of water. The solution ofpectin in water may be obtained by beating for a short period of time (1to 2 minutes in a Waring blendor). Following this, 400 cc. of percentethyl alcohol is added to the solution of pectin and water (equalvolume) This solution of pectin in water plus alcohol is again blendedfor 1 to 2 minutes in aWaring blendor. The pectin'which is insoluble inalcohol is precipitated by the alcohol. To this mixture is added cc. ofsesame oil or peanut oil and the whole is again blended in a Warinblendor for a sufiicient period of time (1 to 3 minutes) to obtain awhite emulsion of oil and water and alcohol with'pectin. At this point asmall quantity of the material examined microscopically for the presenceof particulate pectin shows particle size to be uniformly from 0.1 to1.0 micron in diameter. Since in this process the pectin gel is beingtransformed from a solid solution to particulate pectin (in other wordsbeing precipitated), it is necessary to make a microscopic examinationto be assured that all of the pectin in solid solution has been carriedto the colloidal suspension state. If on the first microscopicexamination, described above, particulate size particle pectin has beenobtained only in part, alcohol (95 percent ethyl alcohol) should beadded followed by further blending in a Waring blendor until suchparticulate pectin is obtained.

The suspension of pectin in alcohol, water and sesame oil is now placedin a, large distilling flask under vacuum and completely deaerated. Thedistilling apparatus, still under vacuum, is placed in a water bath at90 C. and distillation continued at approximately this temperature (85to 95 C.) until all alcohol and water is removed. The final suspensionof pectin in 011 should contain not more than 1 percent moisture. Duringthe distillation process, many of the fine pectin particles aggregate inclumps. This, however, is not a deterrent to the final preparation,since the pectin suspension in oil may be later homogenized by use of aWaring blendor or other similar apparatus.

After the moisture and alcohol have been completely removed from thepectin in oil suspension, the suspension is vigorously homogenized toproduce an even, smooth suspension of pectin in oil. For each 200milligrams of the pectin utilized in the preparation of the aboveparticulate size pectin in oil, is added milligrams of sodiumcrystalline penicillin (plus 10 percent overage). This combination ofpenicillin and pectin in oil is then vigorously milled to form a uniformsusof water. I

be obtained by beating for a short period of time pension. The finalproduct is then filled aseptically into containers for clinical use.This suspension does not settle out on standing.

A second method for production of this particle size pectin eliminatingalcohol is as follows:

Twenty grams of pectin is dissolved in 400 cc. Solution of the pectin inwater may (1 to 2 minutes in a Waring blendor). To this solution isadded 100 cc. of sesame or peanut oil and the whole is again blended in'a Waring blendor for a sufiicient period of time (1 to 3 minutes) toobtain a white emulsion of oiland water'with pectin. At this point asmall quantity of the material examined microscopically for the presenceof particulate pectin will show particles uniformly from 0.1 to1.0'micron in diameter. Here again, since in this process the pectin gelis being transformed from. a solid solution to particulate pectin, it isnecessary to make a microscopic examination to be assured thatall of thepectin in solid solution has been carried to the colloidal suspensionstate. If on the first microscopic examination, described above,particulate size particle pectin has been obtained only in part, furthermilling of the suspension should be carried out until all of the gel"has been transformed to a colloidal suspension of pectin. Thissuspension is placed in a large distilling flask under vacuum,deaerated, and the water removed. The final suspension should containnot more than 1 percent moisture. The process then follows as in theabove case.

In the above I have described procedures for securing stability. As Ihave stated, stability is also achieved by a procedure usinghydrogenated oil. The preferred method in the preparation of such pectinand penicillin in oil is as follows:

For each 300,000 units of penicillin (approximately 180 mg. of sodiumpenicillin and 162 mg. of potassium penicillin), 200 to 300 mg. ofpectin (200 grade or better) is prepared. (I prefer to to use 270 mg.)The pectin must meet the requirements of the National Formulary.

Amounts less than 200 mg. of pectin tend to make a product lesseflicacious in prolonging blood concentrations of this drug and over 300mg. of pectin tends to make the product so viscous that diflicult is hadin preparation and injection of the product. The 2'70 mg. of pectin pereach 300,000 units of penicillin is prepared by drying at a temperatureof 60 to 100 C., being careful not to overheat the pectin since thiscauses discoloration and loss of its gel properties.

I prefer to heat at 100 C. intermittently for 2 to 3 hours at a timeuntil the moisture content is reduced to 1% or less. Under controlledhumidity of relative humidity or less, the pectin is ground in a hammermill or micropulverizer and passed through a 250 mesh screen to obtain aparticle size of approximately 50 microns.

Grinding some types of pectin can in this case also be facilitated bythe addition of small pieces of Dry Ice 11" in diameter) chilling for Aof an hour followed by grinding Dry Ice and pectin together. Thechilling in this case causes the pectin to become brittle and to grindmore readily. The carbon dioxide and any residual moisture must beremoved by vacuum drying. I prefer to use a vacuum oven at 60 C. and arelatively high negative pressure (approximately 100 microns ofmercury).

, The penicillin used is dried to a moisture content of 1% or less andpassed through a 140 mesh screen to obtain a particle size ofapproximately microns. Larger particle size penicillin would be moreeflicacious but here again as with the pectin, too large particle sizecauses difliculty in handling and in passage through relatively smallbore needles (i. e., 20 gauge).

The two dry ingredients (pectin and penicillin) are milled together,always maintaining the above specified moisture content of 1% or less tomake a homogeneous mixture and to this dry mixture is added sufllcientsesame oil or peanut oil (other vegetable oils may be used), the latteroils containing from 5% to 10% hydrogenated peanut oil by volume to makea total quantity of 1 cc. for each 270 mg. of pectin plus 300,000 unitsof penicillin.

Other hydrogenated oils may be used also in place of hydrogenated peanutoil.

Inasmuch as the hydrogenated oils are solid, it is necessary to heatthese oils to about 60 C. to melt and furthermore to heat the vegetableoil to which it is added to approximately the same temperature beforeaddition to the dry ingredients (pectin and penicillin). Moreover, thetemperature should be maintained during the filling operations into thesyringes or cartridges from which the product is ejected.

Aseptic technique must be utilized throughout the preparation of theproduct to ensure its sterility. Both the vegetable oil used and thehydrogenated vegetable oil used must be relatively free from free acids(.05% or less) and have moisture contents of 1% or less.

Since the desired preparation for injection has a volume of 1 cc. andcontains 300,000 units of penicillin, there are a number of factors thatmaterially influence the making of the product. Thus, for example, ifcrystalline sodium penicillin is used in the product, mg. are necessaryto make 300,000 units, while if crystalline potassium penicillin isused, only approximately 162 mg. are needed.

On the other hand, if amorphous penicillin is used (c. g., calciumpenicillin) the weight will depend on its purity; thus if suchpenicillin has a purity or potency of 1000 units per mg. (and this is anormal potency commercially) 300 mg. would have to be used in each 1cc.,to give 300,000 units "per cc. Thus with amorphous penicillin thebulk of the penicillin itself increases the viscosity of the preparationand thus tends to regulate in part at least the quantities of pectin,oil and hydrogenated oil in the final mixture.

If in the preparation of penicillin in oil and pectin, 300,000 units ofamorphous calcium penicillin having a potency of 1000 units per mg. isused in a total volume of 1 cc. a total of 300 mg. of the penicillin isnecessary. Since 270 mg. of pectin is preferred, and this in itselfeflects the viscosity of the final preparation, it has been found thate. g. 10% hydrogenated peanut oil cannot be utilized in such apreparation because the viscosity is so great that difficulty isencountered on injection. (The product becomes a stiff paste that cannotbe handled by the clinician.)

Thus when amorphous penicillin is used, it is necessary to use from 5%to 7.5% hydrogenated vegetable oil for stabilization purposes.

When crystalline penicillin is used in the preparation less diflicultyis encounteredsince two factors are relatively fixed; i. e., the volume(1 cc.) and the weight of penicillin 162-180 mg. Thus it is possible toregulate the amount of pectin and hydrogenated oil within limits.

Both pectin I and hydrogenated oils tend to prolong blood concentrationsof penicillin although the latter was added to the preparation initiallyto increase the stability of the product, i. e., to prevent the settlingof the pectin.

From experiments it has been shown that 4% or less of the hydrogenatedpeanut oil does not prevent settling of the pectin; thus the lower limitis fixed at 5%. Increasing quantities of this hydrogenated oil increasesthe viscosity so that at a 15% concentration in the presence of 200 mg.of pectin and 180 mg. of crystalline potassium penicillin, greatdifllculty is encountered in-injecting the preparation. It may .beassumed therefore that the upper limit of concentration of thehydrogenated peanut oil under these conditions is approximately 15%.

Because the success of the preparation is de pendent on the formation ofa gel with pectin in the muscle tissue utilizing the water from suchtissue to form the gel, the greater the amount of pectin utilized, thestiffer the gel and the more prolonged the blood concentrations ofpenicillin entrapped in the gel. This has been taken into considerationin reaching a decision as to the quantities of pectin and hydrogenatedoil to be utilized with a fixed amount of penicillin.

Thus to produce the most satisfactory product, the optimum amount ofpectin should be used and this appears to be 270 mg. per cc. ofpreparation. However, suflicient hydrogenated oil must 'be added toprevent settling and although 5% will be satisfactory, the optimumamount appears to be from 7.5% to In order to determine the effect ofpectin on injection into the muscles of rabbits, three normal adultrabbits were injected intramuscularly in the right rear legs with l nil.of a suspension containing 300,000 units of potassium penicillin, 270mg. of high-grade pectin, and sufficient sesame oil to make 1 ml. After24 hours, palpa tion of the muscles at the site of injection showed noindication of nodules. The animals were sacrificed and the musclesinjected were excised and observed grossly for evidence of residualpectin and oil, and for evidence of inflammation. At the insertion ofthe hypodermic needles in the muscles, the usual traumatic injury wasevident on gross examination. Further evidence of tissue damage was notobserved. Deposits of oil were found but residual pectin could not bedemonstrated. The picture observed is that which is found on injectionof 1 ml. of sesame oil alone.

The same suspension was injected subcutaneously into two rabbits, onereceiving the injection on the shaven back surface and the other on asimilarly prepared area of the abdomen. Palpation in these areas 24hours later showed no evidence of nodules. The rabbits were sacrificedand autopsied. Gross observation of the tissues into which the injectionhad been made revealed no abnormalities.

Since in no instance was it possible to demonother area with a controlsuspension of the dye in sesame oil alone. Twenty-four hours later, allthree animals were examined for evidences of nodule deposits of pectin.None were found. The three animals were sacrificed and the areas intowhich the injections had been made were located by tracing the dye.These muscle areas were removed and placed in 70% alcohol forpathological study. 5 I

Grossly, there appeared to belittle or no irritation other than thatapparently produced by the dye itself. In two animals, hemorrhagic areaswere noted-at the point of injection. These appeared to .be a traumatici jury, resulting from the needle itself. Oil deposits were found but inno instance could residual pectin deposits be demonstrated. A comparisonof the areas of the muscles injected with the dye alone with those areasof the muscle injected with the dye plus pectin indicated that lessinjury occurred with the combination of pectin and dye than with the dyealone. This was quite evident after the muscles had been fixed informalin for paraifin sectioning. This result may be explained. on abasis that the dye itself in the pectin preparation was slowly releasedfrom the repository site of injection.

In the above I have described the use of pectin penicillin. As Ihavealready stated and as outlined in the parent application, the pectinpreparation with other pharmaceutical products also produce prolongationof therapeutic effect. Thus the pectin or other similar hydrophilicproduct described in the parent application may be used with otherantibiotics such as streptomycin, derived from streptomyces griseus;penicillin X, penicillin F, penicillin G, penicillin dihydro F,penicillin K, derived from various strains of penicillin notatum, andpenicillin chrysogenum; bacitratcin, eumycin and 'subtilin, derived fromB. subtilis and other similar mold or bacterial excretory productspossessing antibiotic properties.

Both the mold and the bacterial excretory products may be in the form ofsalts such as penicillin sodium or penicillin calcium, streptomycinsulphate, streptomycin phosphate, streptomycin strate the presence ofpectin following both subcutaneous and intramuscular injections, itseemed advisable to do further studies in which the injection would betraced by simultaneous injection of a dye. In these experiments asuspension of pectin (235 grade) was prepared in refined sesame oil andto it added .5% tartrazine (F. D. C. yellow). For a control, .5%solutions of tartrazine were prepared in sesame oil only. The, rear legsof three rabbits were depilated and an injection consisting of 1 ml. ofthe pectin suspension containing the dye was injected intramuscularlyinto each leg. Each muscle was also injected in anhydrochloride,streptomycin calcium chloride, trihydrochloride double salt, etc.

Drugs such as insulin, epinephrine, ephedrine and hormones, includingestrin, estradiole and stilbesterol; vitamins such as folic acid,vitamin B1 complex, vitamin C (ascorbic acid), liver extract and drugswhere the therapeutic effect is to be prolonged, can also be made andused in accordance with this invention.

Thus in the case of streptomycin, 1 cc. of streptomycin pectin in oilconsistsof from 160 to 200 mg. (preferably 160 mg.) of pectin of aparticle size of 50' microns (passing through a 250 mesh screen)containing less than 1% moisture and 400 to 500 mg. of activity ofstreptomycin of micron particle size and containing less than 1%moisture.

In streptomycin, one half gram (500 mg.) of activity is actuallyequivalent to'about 800 mg. of material. This is due to the fact thatstreptomycin base has an activity of 1000 micrograms per mg. and whenthe base is made into the salts which are streptomycin phosphate,streptomycin hydrochloride, streptomycin sulphate or streptomycincalcium chloride trihydrochloride (double salt) the added weight of theacids utilized in their preparation decreases the activity per mg.

Ideally for clinical reasons, /2 gram of activity per injection in 1 cc.should be used. Thus if approximately 800 mg. of a streptomycin salt(only 500 of which is active) is used and to this is added 200 mg. ofpectin, there is already a total of 1000 mg. (1000 mg. of water is 1cc.) to which there still must be added oil and hydrogenated oil. Thisis one reason that the preparation as described hereinafter hasapproximately 800 mg. of streptomycin, 160 mg. of pectin, and sufllcientsesame oil to make 1 cc., the sesame oil containing 5% hydrogenatedpeanut oil.

The pectin used in this preparation is prepared in the same manner asthat used in penicillin in pectin and oil, i. e., ground to properparticle size, sterilized by dry heat, and dried to a moisture contentof 1% or less. The streptomycin is passed through a 140 mesh screen toobtain a particle size of approximately 100 microns, dried to a.moisture content of 1% or less, and mixed thoroughly with the pectin.For each 0.4 to 0.5 gram of streptomycin is added 160mg. of the abovepectin. Sesame oil is heated to about 60 C. and to it is added 5% byvolume of hydrogenated peanut oil at the same temperature. Maintainingapproximately this temperature and for each 0.4 to 0.5 gram ofstreptomycin plus 160 mg. of pectin, there is' added sufliclent of thesesame oil containing hydrogenated peanut oil to make The pectinstreptomycin and oils are thoroughly blended in a Waring blendor orsimilar device and while still at a temperature of approximately 60 C.filled asceptically into cartridges or syringes. Utilizing a preparationof streptomycin in pectin and 01] prepared as described above, eighteenpatients were treated with 1 cc. each (.42 gram of streptomycin). Bloodsamples were taken from six patients during the first 8 hours, from sixpatients from the 12th to 24th hours, and from six patients from the thto 48th hours and the samples assayed by the B. circulans serialdilution method for their streptomycin-content. (The samples were takenin the above manner to avoid bleeding each patient a large number oftimes). The results obtained are given in the following table where itwill be noted that all patients tested had therapeutic concentrations ofstreptomycin in their blood 48 hours after a single injection of 0.42gram in the pectin preparation. (It should be pointed out that incontrast to the Pectin preparation, a preparation of streptomycinbeeswax and oil shortens the activity of streptomycin in the body ratherthan prolongs it. As a matter of fact, aqueous solutions of streptomycingive a more prolonged therapeutic concentration in the body than dosimilar amounts of streptomycin in beeswax and oil.)

TABLE SHOWING BLOOD CONCENTRATIONS OF STREPTOMYCIN FOLLOWING A SIN- GLEINJECTION OF .42 GRAM IN PECTIN AND OIL CONTAINING 5% HYDROGEN- ATEDPEANUT OIL Name 12 hr 15 hr 20 hr. 24 hr Name hr 36 hr 42115. 48 hr Fromthe physical appearance of streptomycin-pectin in oil it seemed that acompound might possibly be formed in the presence of the water of thebody tissues since streptomycin is a base and pectin an acid. Such acompound would release the streptomycin as the body broke down thepectin. On this basis a gram of pectin and a gram of streptomycin wereplaced in solution and allowed to stand one hour. On mixing some heatwas evolved. The material was then treated with 3 volumes of ethylalcohol to precipitate the pectin. A gelatinous material wasprecipitated and approximately 75% of the streptomycin was found toprecipitate with it. This would indicate that either the streptomycinwas physically bound or chemically bound to the pectin.

To test the effectiveness of the pectin-streptomycin in aqueous solutionin prolonging the action of streptomycin, nine rabbits were injectedeach with 300,000 micrograms of the solution in a 1 cc. volume. Twokinds of streptomycin were used (crystalline and amorphous) and twoconcentrations of pectin 2.5% and 1.25%.

The following table shows the results obtained:

TABLE I Streptomycin Pectin solution, potency 300,000 mcg. per CC.

[Micrograms per cc. of blood serum] Material Injected Hours afterinjectlon- 18 hrs. 24 hrs.

Crystalline Streptomycin plus 1. 1. 4

1.25% pectin. 1.65 0. 79 0.26 0. 25

Average level 1. 15 81 Crystalline Streptomycin plus 1. 27 .545 2.5%pectin. 1. 33 600 1.07 395 Average level 1. 22 51 Amorphous Streptomycinplioa- R 3.20 2. 10 phate plus 1.25% pectin. IS 2. 00 0. 88 IT l. 250.89

Average level 2. 15 1. 29

TABLEIII Streptomycin only, potency 300,000 mcg. per cc.

micrograms per cc. of blood serum] Hours after injection 24 hrs.

Rabbit f Rabbit f2 Rabbit #8 Average level.

is sea-assess t: as. see

TWO RABBITS INIECTED WITH 000,000 MCG.

Rabbit l 80 Rabbit #10 1. 75 1. 05

Average leveL l. 73 93 It will be noted that the average level obtainedwith streptomycin alone was considerably less at both 18 and 24 hoursthan the levels obtained with the streptomycin-pectin combination inaqueous solution. 1

On the basis of the results obtained in animals, human experiments wereinitiated using three types of streptomycin, i. e., streptomycinsulphate, streptomycin hydrochloride and streptomycin phosphate. Thestreptomycin salts were prepared as follows:

A 1.25% solution of pectin was prepared by adding for each 100 cc. ofdistilled water 1.25 grams of pectin. This was placed in solution bywhirling in a, Waring blendor for fifteen minutes. The solution ofpectin in water was then autoclaved under a steam pressure of pounds(120 C.) for twenty minutes for sterilization. After cooling to roomtemperature, each of the three salts was added to the pectin solution tomake a concentration of 0.4 gram of streptomycin activity in each onecc. of pectin solution.

A considerable amount of heat was evolved on mixing the streptomycinwith the pectin solution. The heat evolved, apparently one of chemicalinter-reaction between the pectin and streptomycin, appeared to begreatest with streptomycin phosphate. Thefact that heat is evolved onmixing these substances, that on precipitation with alcohol of thepectin, streptomycin is also precipitated (alcohol will not precipitatestreptomycin from solution) and prolongation of streptomycin activity isobtained in the animal body with this preparation, all oifer evidence tosupport the idea that a chemical compound is obtained on mixing inaqueous solution pectin and streptomycin.

"The preparation described above was then added aseptically to cc. vialsfor dispensing and checked for sterility. After determining that theproduct was sterile, a group of patients were injected at the clinicwith this material in a dose of 1.0 cc. per patient and blood samplestaken at intervals to determine whether a prolongation of activity couldbe obtained. The results are shown in Table III.

TABLE III SrimP'ronYcm, 0.4 on. PER Pin-rm 0-20-17 Streptomycin 400,000meg/ml. aqueous tin 12.5 m mlthree salts of streptomycin and two math 5of assay use [Micrograms per cc. of blood serum.}

8. GIRGULANS SERIAL DILU'IION 12hour lohour mhour zihour 1 Too low.POP-Streptomycin phosphate.

HCl-Streptomycin h drochloride. S04Streptomycinsu am.

It will be noted from the table that all patients injected withapproximately 400,000 micrograms (.4 of a gram) of streptomycin-pectinin solution, whether the salt was the phosphate, hydrochloride or thesulfate, showed streptomycin in their blood stream twenty-four hoursafter injection. Thus, it is possible with this preparation to obtaintherapeutic levels of streptomycin in the blood for this period thusobviating the necessity for frequent injections during the day.

Several points are listed below concerning this preparation 1.Streptomycin-pectin in solution in water forms a clear, easilyinjectable solution and may be injected with any type of syringe.

2. The evidence appears to indicate that the streptomycin-pectin insolution forms a compound under the conditions set up herein for itspreparation.

3. Although 1.25% and 2.5% pectin have been utilized, 5%, 10%, and 15%solutions have been prepared by proper treatment with steam underpressure. A 5% concentration of pectin in solution is an extremely stiffgelatinous mass which cannot be injected as such, but on treatment withsteam under pressure (autoclaved) the pectin molecule is reduced in sizeand a treatment consisting of steam pressure of fifteen pounds fortwenty minutes causes a 5% heavy gelatinous mass to become liquid andthus utilizable in a syringe.

4. In view of the fact that streptomycin is a base and pectin is anacid, the steam treatment of pectin and reduction in size of themolecule resultsin the formation of more radicals available forattachment to the streptomycin. It is felt that within limits the morepectin that can 13 be utilized in this preparation the more efllcientthe preparation will be in prolonging the blood levels of streptomycin.

5. Streptomycin-pectin in solution may be prepared in multiple-dosecontainers for dispensing by physicians.

6. The preparation of streptomycin-pectin in solution is extremelysimple since it entails autoclaving the pectin in water solution for adefinite period of time followed by the addition aseptically of one ofthe streptomycin salts.

7. Preliminary stability data carried out at temperatures of 25 C., 37C., and 56 C. indicate that the product is stable and does not losepotency over a period of at least one week at a 1 temperature as high as56 C.

8. If it is found that the stability of this preparation is not as greatas anticipated, the preparation could be prepared in a so-calledcombination package. Such a package would consist of a solution, e. g.,of pectin sterilized by treatment in the autoclave in one vial and onegram of streptomycin in the second vial. The physician would then mixthe two vials before use by placing the pectin solution in the drystreptomycin salt aseptically with a sterile syringe.

9. It should be noted in Tables I and II that the streptomycin-pectin inaqueous solution in which 300,000 micrograms of streptomycin wasincorporated gave blood levels in rabbits of approximately the sameheight as 600,000 micrograms of streptomycin alone.

The invention described herein may be manufactured and used by or forthe Government of the United States for governmental purposes withoutthe payment to me of any royalty thereon in accordance with theprovisions of the Act of April 30, 1928 (Ch. 460, 45 Stat. L. 467).

I claim:

1. An injectable dry therapeutic composition comprising the combinationof an innocuous oil having colloidal pectin dispersed therein, with asolid, finely-divided drug of a particle size of about 50 to 100 micronsnormally tending to settle out of suspension, and hydrogenated peanutoil as a suspension stabilizer.

2. An injectable dry therapeutic composition 14 comprisingthecombination of peanut oil having colloidal pectin dispersed therein,with asolid, finely-divided penicillin of a particle size of about tomicrons normally tending to settle out of suspension, and hydrogenatedpeanut oil as a suspension stabilizer.

3. An injectable dry therapeutic composition comprising the combinationof peanut oil having colloidal pectin dispersed therein, withsolid,finely-divided streptomycin of a particle size of about 50 to 100microns normally tending to settle out of suspension, and hydrogenatedpeanut oil as a suspension stabilizer.

4. An injectable dry therapeutic composition consisting of, per cubiccentimeter, the combination of a peanut oil vehicle having about 2'70mg. of colloidal pectin dispersed therein with about 300,000 units offinely-divided penicillin having a particle size of about 50 to 100microns normally tendin to settle out, and between five and ten percentof hydrogenated peanut oil as a suspension stabilizer.

HENRY WELCH.

REFERENCES CITED The following references are of record in the tile ofthis patent:

UNITED STATES PATENTS Number Name Date 2,055,083 Klein Sept. 22, 19362,413,419 Saunders Dec. 31, 1946 OTHER REFERENCES Science, Sept. 1,1944, pages 196 to 198.

Zimmamon, Venereal Disease Information, February 1945, page 31.

Lloyd Jones, The Lancet, April 13, 1946, page 526.

Raiziss, Science, November 3, 1944, pages 412 and 413.

Hobby et al., Proc. Soc. Exptl. Biol. 8: Med., June 1942, page 286.

Science News Letter, October 4, 1947, page 210.

J. Amer. Pharm. Assoc., Sci. Ed., Jan. 1948, pages 21 to 23.

4. AN INJECTABLE DRY THERAPEUTIC COMPOSITION CONSISTING OF PER CUBICCENTIMETER, THE COMBINATION OF A PEANUT OIL VEHICLE HAVING ABOUT 270 MG.OF COLLOIDAL PACTIN DISPERSED THEREIN WITH ABOUT 300,000 UNITS OFFINELY-DIVIDED PENICILLIN HAVING A PARTICLE SIZE OF ABOUT 50 TO 100MICRONS NORMALLY TENDING TO SETTLE OUT, AND BETWEEN FIVE AND TEN PERCENTOF HYDROGENATED PEANUT OIL AS A SUSPENSION STABILIZER.